The invention relates to new vitamin D analogs, to methods of preparing these compounds and to their use in pharmacotherapy and cosmetics.

It is generally known, that vitamin-D compounds or vitamin-D related compounds ("vitamin-D analogs") have a strong biological activity and may be used in all those cases in which problems with the calcium and bone metabolism play a part. A few years ago it was found that various active vitamin-D compounds and analogs also have other pharmacotherapeutic activities and may be used successfully, for example, for the treatment of certain skin and bone diseases, for cosmetic applications and for treating diseases which are related to cell differentiation, cell proliferation or imbalance in the immune system, including diabetes mellitus, hypertension and inflammatory diseases such as rheumatoid arthritis and asthma. In addition, these compounds may be used in various veterinary applications, and for diagnostic purposes.

Further it is known from J. Med. Chem. 38 (1995), 4529-4537 that 1 α, 25-dihydroxy vitamin D₃ analogs of the formula wherein S₁ is OH, HOCH₂- or HOCH₂CH₂ have very low affinities, i.e. no more than 10^-3 the affinity of calcitriol, for the calf thymus vitamin D receptor, and considerable antiproliferative activities in murine keratinocytes.

Surprisingly it has now been found that a group of new vitamin D analogs has an activity of up to 6 times that of calcitriol, i.e. is much more active than the above mentioned known analogs, in the same test (affinity for the calf thymus vitamin D receptor).

The present invention relates to compounds of the general formula wherein

• R₁ is a hydrogen atom or a substituent selected from the group consisting of OH, CH₂OH, CH₂CH₂OH, CH₂CH₂CH₂OH, OCH₃, OCH₂OH, OCH₂CH₂OH and OCH₂CH₂CH₂OH;
• R₂ is a hydrogen atom or a substituent selected from the group consisting of OCH₃, OCH₂OH, CH_m(CH₂OH)_n(CH₂CH₂OH)_p, (CH₂)_qOH and O(CH₂)_rOH;
     wherein:
        m is 0 or 1, p, q and n are 0-3, r is 1-3 and
        m+n+p=3;
  with the proviso that R₁ and/or R₂ contain at least one OH group;
• R₃ is a straight or branched, saturated or unsaturated aliphatic hydrocarbon of 6-13 C atoms which may be substituted with one or more substituents from the group hydroxy or fluoro.

The above new vitamin D analogs of the invention, presented by the general formula I, are valuable substances. The biological results, as illustrated in the Examples, indicate that these compounds are promising as biologically active substances and may be used in all above-mentioned pharmacotherapeutic indications, more in particular for the treatment of osteoporosis, renal osteodystrophy, osteomalacia, skin disorders such as psoriasis (and other hyperproliferative skin diseases), eczema and dermatitis, myopathy, leukaemia, breast and colon cancer, osteosarcomas, squamous cell carcinomas, melanoma, certain immunological disorders, and transplant rejections.

Furthermore, the new vitamin D analogs of the invention may be used for wound healing and may be incorporated in cosmetic compositions, such as creams, lotions, ointments and the like, in order to preserve, condition and/or protect the skin and to improve various skin conditions, such as wrinkles, dry skin, skin slackness and insufficient sebum secretion. The new vitamin D analogs may also be used for diagnostic purposes.

Preferred compounds of the general formula (I) are the compounds wherein R₃ is the group-(CH₂)₃-C(CH₃)₂OH.

Especially preferred are the compounds of formula (I) wherein R₃ is the group-(CH₂)₃-C(CH₃)₂OH and wherein:

• a) R₁ is a hydrogen atom and R₂ represents CH₂CH₂OH, CH₂CH₂CH₂OH, CH(CH₂CH₂OH)₂, C(CH₂CH₂OH)₃ or C(CH₂CH₂OH)₂CH₂OH; or
• b) R₁ is OH, CH₂OH or (CH₂)₂OH and R₂ represents hydrogen, OH, OCH₃, or O(CH₂)_rOH, wherein r has the value 1 to 3.

Most particularly preferred are the compounds of formula (I) wherein R₃ is the group -(CH₂)₃-C(CH₃)₂OH and wherein:
   R₁ is hydroxy, CH₂OH and CH₂CH₂OH and R₂ represents hydrogen, CH₂OH or CH₂CH₂OH.

It is a special merit of the present invention that the above new vitamin D analogs can easily be prepared from readily available starting materials.

Consequently, the invention also relates to a method of preparing a vitamin D analog of the general formula I, as defined above, which method is characterized in that a compound of the general formula wherein:

• R₃' is a straight or branched, saturated or unsaturated aliphatic hydrocarbon of 6-13 C-atoms which may be substituted with one or more substituents from the group protected hydroxy or fluoro, is reacted with an organometallic compound of the general formula
• R₁' and R₂' are the hydroxy protected analogs of R₁ and R₂ defined above, X is Li,MgCl, MgBr or MgI, to yield a compound of the general formula

The compounds having formula I can also be obtained by reacting a compound of the general formula wherein R₃' has the meaning given above, and R₄ is a protected hydroxy group, with a compound of the general formula wherein X' is halogen, preferably Br, and R₁' and R₂' have the above given meanings, under the influence of an alkyl lithium compound, a zinc halogenide, preferably ZnCl₂, and a transmetallation catalyst, followed by deprotection of the hydroxy group(s). An example of a transmetallation catalyst is tris-(dibenzylidene-acetone)-dipalladium(0).

The enolic hydroxy group of enolized compound IV is preferably derivatized by a reaction with N-aryltriflimide to produce a triflate.

To improve the applicability of the new vitamin D analogs of the invention for the above-described pharmacotherapeutic indications, the compounds are usually processed to pharmaceutical compositions, comprising an effective amount of said vitamin D analog as the active ingredient in addition to a pharmaceutically acceptable carrier and/or at least one pharmaceutically acceptable auxiliary substance. Such a composition may be delivered in a dosage unit form for oral, topical (dermal) or parenteral administration, comprising approx. 0.1 µg to approx. 0.1 mg active ingredient per dosage unit.
A composition for diagnostic purposes may comprise, in addition to the vitamin D analog of the present invention, a compatible, non-toxic carrier and/or at least one auxiliary substance.
A cosmetical composition may comprise, in addition to an effective amount (in the range of approx. 0.1 µg to approx. 0.1 mg per dosage unit in a dosage unit form) of the vitamin D analog of the present invention, a cosmetically acceptable, non-toxic carrier and/or at least one auxiliary substance.

Finally the invention relates to a method for the treatment and prophylaxis of a number of disease states including autoiummune diseases (including diabetes mellitus), acne, alopecia, skin aging (including photo-aging), imbalance in the immune system, inflammatory diseases such as rheumatoid arthritis and asthma, as well as diseases related to abnormal cell differentiation and/or proliferation, in a warm-blooded living being, comprising administering to said being or treating said being with a pharmaceutical composition as defined above in a quantity effective for the intended purpose. Examples of such diseases are psoriasis and other hyperproliferative skin diseases.
The present invention also relates to the use of the above pharmaceutical compositions for the treatment of solid, skin and blood cancers, in particular of blood cancers such as leukaemia, of breast and colon cancer, and of skin cancers such as melanoma and squamous cell carcinoma.
The above-defined cosmetical compositions, in particular selected from the group consisting of creams, lotions, ointments, liposomes and gels, can be used for the treatment and prevention of a number of skin disorders, such as inadequate skin firmness or texture, insufficient skin hydration, wrinkles and insufficient sebum secretion.

The invention will now be described in greater detail with reference to the following specific Examples.

The following abbreviations are used in the examples:

• THF = tetrahydrofuran
• TBDMS = tert.-butyl dimethyl silyl
• DMF = N,N-dimethylformamide
• Pd₂dba₃ = tris-(dibenzylideneacetone)dipalladium (0)

The TBDMS protected compound (IIIa) wherein X' = Br is dissolved in dry THF, cooled to -78°C and treated with 2 eq. of tert. C₄H₉Li. After 30 minutes a solution of 1 eq. ZnCl₂ in dry THF is added and stirring is continued another 15 minutes. A solution of 1.1 eq. of a compound IV wherein R₄ is CF₃SO₃-, 5 mol % of the catalyst Pd₂dba₃ and 10 mol % of As₃P in dry DMF is introduced by means of double ended needle technique. The mixture is stirred at room temperature until the catalyst has completely dissolved. The progress of the reaction is monitored by thin-layer-chromatography. The reaction mixture is worked up by flash-chromatography. Complete deprotection of the hydroxy groups is carried out by treatment of the obtained product with an ion exchange resin, such as DOWEX-50 W, in methanol.

The following compounds of formula (I) have been prepared in this manner: Comp. No . R₁ R₂ R₃ Yield (%) 1 CH₂CH₂OH H (CH₂)₃-C(CH₃)₂OH 79 2 OH H "         " 51 3 H CH₂OH "         " 82 4 H CH₂CH₂OH "         " 88 5 CH₂OH H "         " 57

¹H-NMR data (CDCl₃,δ) of compounds no.s 1-5: 400 mHz ¹H-NMR (CDCl₃,δ): 0.76 (C₁₈H₃; 3H; s); 1.00 (C₂₁H₃; 3H, d); 1.22 (C₂₆,₂₇H₃, 6H; s); 2.27 (C₁₁H₂, 2H, b); 2.58 (C₁₄H; 1 H, b); 2.83 (Φ-CH₂ - C, 2x; 4H, t); 3.85 (2x Φ-C-CH₂-O, 4H, t); 5.64 (C₉H; 1H, b); 6.92 (A, 1H, s); 6.93 (B; 2H, s). 400 mHz ¹H-NMR (CDCl₃, δ): 0.73 C₁₈H₃; 3H,s); 0.99 (C₂₁H₃, 3H, d); 1.19 (C_26,27H₃; 6H,s); 2.23 (C₁₁H₂, 2H, b); 2.48 (C₁₄H, 1H, b); 5.58 (C₉H, 1H, b); 6.16 (B, 2H, s); 6.20 (A, 1H, s); 8.53 (2xΦ-OH, 2H, s). 400 mHz ¹H-NMR (CDCl₃, δ): 0.77 (C₁₈H₃; 3H, s); 1.00 (C₂₁H₃, 3H, d) ; 1.21 (C_26,27H₃; 6H, s); 2.58 (C₁₄H, 1H, b) ; 4.65 (Φ-CH₂-O, 2H, s); 5.63 (C₉H; 1H, b); 7.18 (Φ-H, 2H, t); 7.26 (Φ-H, 2H, b). 400 mHz ¹H-NMR (CDCl₃, δ): 0.76 (C₁₈H₃, 3H, s); 1.00 (C₂₁H₃; 3H, d);
1.22 (C_26,27H₃, 6H, s); 2.60 (C₁₄H, 1H, b); 2.84 (Φ-CH₂-C, 2H, t); 3.85 (Φ-C-CH₂-O, 2H, t); 5.62 (C₉H, 1 H, b); 7.14 (Φ-H, 4H, b). 400 mHz ¹H-NMR (CDCl₃, δ): 0.76 (C₁₈H₃, 3H, s); 1.00 (C₂₁H₃, 3H, d) ; 1.22 (C_26,27 H₃, 6H, s); 2.59 (C₁₄H, 1H, b) ; 4.64 (2x Φ-CH₂-O, 4H, s); 5.65 (C₉H; 1H, b); 7.11 (2x B; 2H, s); 7.18 (A, 1H; s).

The aromatic vitamin D analogs were dissolved in ethanol in concentrations ranging from 10^-14 to 10^-6 M. The affinity towards the calf thymus intracellular vitamin D receptor (VDR) was determined in a biological in vitro assay and compared to that of calcitriol. In this assay, labelled ³H-calcitriol, which is specifically bound to the VDR, is replaced by the aromatic vitamin D analog. Compounds 5 and 3 have even higher affinities (resp. 6 and 3 times) than calcitriol itself towards the highly selective VDR. Also analogs 4 and 1 demonstrate high VDR affinities. A high VDR affinity is indicative for biologically active substances.

The compounds with the highest affinities were therefore further tested in vitro.

Vitamin D binding protein (DBP) is the specific carrier for vitamin D and its metabolites in blood. The biological activity of vitamin D analogs depends on their binding to DBP. Strong binders will have reduced access to the VDR. Weak binders are rapidly metabolized, which is a favourable aspect in topical application.

DBP is purified from total human serum. In the assay, DBP is incubated with ³H-calcitriol and calcitriol or one of the aromatic vitamin D analogs. To this purpose, the vitamin D analogs are dissolved in ethanol in concentrations ranging from 10^-11 to 2.5 X 10^-6.
The percentage bound/unbound ³H-calcitriol is then calculated. Compounds 5, 4 and 3 (in order of binding) are relatively good binders, binding a factor 2-10 less than calcitriol itself. These binding capacities show that the compounds might have systemic actions, i.e. they might be active when administrated orally or given by injection.